Project Summary Parkinson's disease (PD) is a progressive neurodegenerative movement disorder caused primarily by the degeneration of dopaminergic neurons in the substantia nigra. Current therapies for PD are palliative but no disease-modifying therapies exist today. Mutations in the VPS35 (PARK17) gene have been identified as a cause of late-onset, autosomal dominant PD, with a single mutation (D620N) detected in PD individuals and families worldwide. How mutations in VPS35 precipitate dopaminergic neurodegeneration in PD remains obscure. It is critical to identify the molecular and cellular mechanisms that lead to neurodegeneration due to VPS35 mutations in order to understand the pathophysiology of PD and develop new therapeutic strategies. VPS35 is a core component of the retromer complex responsible for the recognition and sorting of transmembrane protein cargo from endosomes to the Golgi network or plasma membrane for recycling. How familial mutations influence VPS35 retromer function in PD-relevant neuronal populations and animal models is not known. We have developed a novel viral-mediated gene transfer model of PD in adult rats where the overexpression of human D620N VPS35 induces the degeneration of nigral dopaminergic neurons, thereby formally establishing a pathogenic role for the D620N mutation. In the present application, we propose to exploit this disease model to elucidate the full repertoire of PD-related neurodegenerative phenotypes induced by D620N VPS35, including the development and progression of dopaminergic neuronal and axonal degeneration, striatal catecholamine and motoric deficits, neuropathology, protein aggregation and ultrastructural cytopathology, and altered macroautophagy, as a function of increasing transgene dosage (Aim 1.1). We will evaluate perturbations in retromer protein interactions by quantitative proteomic analyses of brain tissue from this model and primary neurons to identify putative molecular mechanisms underlying the D620N mutation, and we will determine whether selected protein interactors are important for neurodegeneration (Aim 1.2). Our studies will also clarify the mechanism of the dominant D620N mutation in this rodent PD model and will address whether D620N VPS35 may act in a dominant-negative manner. Accordingly, we will use viral vectors to deliver short hairpin RNAs to silence VPS35 expression in nigral dopaminergic neurons of rats to create a VPS35 loss- of-function model and conduct rescue experiments with human VPS35 variants to determine whether the D620N mutation is functional or impaired (Aim 2.1). Similar rescue studies will be conducted in new conditional VPS35 knockout mice with selective deletion in neurons (Aim 2.2). Finally, we will evaluate the interaction of VPS35 with LRRK2 in this rat model of PD to determine whether LRRK2 kinase activation is critically required for neurodegeneration induced by D620N VPS35. We will determine if LRRK2 deletion (Aim 3.1) or pharmacological kinase inhibition (Aim 3.2) in this rat model is neuroprotective. Our proposal is novel, innovative and timely and will provide critical insight into the mechanisms of PD-linked VPS35 mutations using transgenic rodent models.